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Neutrophil extracellular traps (NETs) are intricate, DNA-based, web-like structures adorned with cytotoxic proteins. They play a crucial role in antimicrobial defense but are also implicated in autoimmune diseases and tissue injury. The process of NET formation, known as NETosis, is a regulated cell death mechanism that involves the release of these structures and is unique to neutrophils. NETosis is heavily dependent on the production of reactive oxygen species (ROS), which can be generated either through NADPH oxidase (NOX) or mitochondrial pathways, leading to NOX-dependent or NOX-independent NETosis, respectively. Recent research has revealed an intricate interplay between ROS production, DNA repair, and NET formation in different contexts. UV radiation can trigger a combined process of NETosis and apoptosis, known as apoNETosis, driven by mitochondrial ROS and DNA repair. Similarly, in calcium ionophore-induced NETosis, both ROS and DNA repair are key components, but only play a partial role. In the case of bacterial infections, the early stages of DNA repair are pivotal. Interestingly, in serum-free conditions, spontaneous NETosis occurs through NOX-derived ROS, with early-stage DNA repair inhibition halting the process, while late-stage inhibition increases it. The intricate balance between DNA repair processes and ROS production appears to be a critical factor in regulating NET formation, with different pathways being activated depending on the nature of the stimulus. These findings not only deepen our understanding of the mechanisms behind NETosis but also suggest potential therapeutic targets for conditions where NETs contribute to disease pathology.

The complement system and neutrophils play crucial roles in innate immunity. Neutrophils release neutrophil extracellular traps (NETs), which are composed of decondensed DNA entangled with granular contents, as part of their innate immune function. Mechanisms governing complement-mediated NET formation remain unclear. In this study, we tested a two-step NETosis mechanism, as follows: classical complement-mediated neutrophil activation in serum and subsequent NET formation in serum-free conditions, using neutrophils from healthy donors, endothelial cells, and various assays (Fluo-4AM, DHR123, and SYTOX), along with flow cytometry and confocal microscopy. Our findings reveal that classical complement activation on neutrophils upregulated the membrane-anchored complement regulators CD46, CD55, and CD59. Additionally, complement activation increased CD11b on neutrophils, signifying activation and promoting their attachment to endothelial cells. Complement activation induced calcium influx and citrullination of histone 3 (CitH3) in neutrophils. However, CitH3 formation alone was insufficient for NET generation. Importantly, NET formation occurred only when neutrophils were in serum-free conditions. In such environments, neutrophils induced NADPH oxidase-dependent reactive oxygen species (ROS) production, leading to NET formation. Hence, we propose that complement-mediated NET formation involves a two-step process, as follows: complement deposition, neutrophil priming, calcium influx, CitH3 formation, and attachment to endothelial cells in serum. This is followed by NADPH-dependent ROS production and NET completion in serum-free conditions. Understanding this process may unveil treatment targets for pathologies involving complement activation and NET formation.

Neutrophil extracellular traps (NETs) are cytotoxic DNA-protein complexes that play positive and negative roles in combating infection, inflammation, organ damage, autoimmunity, sepsis and cancer. However, NETosis regulatory effects of most of the clinically used drugs are not clearly established. Several recent studies highlight the relevance of NETs in promoting both cancer cell death and metastasis. Here, we screened the NETosis regulatory ability of 126 compounds belonging to 39 classes of drugs commonly used for treating cancer, blood cell disorders and other diseases. Our studies show that anthracyclines (e.g., epirubicin, daunorubicin, doxorubicin, and idarubicin) consistently suppress both NADPH oxidase-dependent and -independent types of NETosis in human neutrophils, ex vivo. The intercalating property of anthracycline may be enough to alter the transcription initiation and lead NETosis inhibition. Notably, the inhibitory doses of anthracyclines neither suppress the production of reactive oxygen species that are necessary for antimicrobial functions nor induce apoptotic cell death in neutrophils. Therefore, anthracyclines are a major class of drug that suppresses NETosis. The dexrazoxane, a cardioprotective agent, used for limiting the side effects of anthracyclines, neither affect NETosis nor alter the ability of anthracyclines to suppress NETosis. Hence, at correct doses, anthracyclines together with dexrazoxane could be considered as a therapeutic candidate drug for suppressing unwanted NETosis in NET-related diseases.

: Neutrophils undergo a unique form of cell death that generates neutrophil extracellular traps (NETs) that may help to neutralize invading pathogens and restore homeostasis. However, uncontrolled NET formation (NETosis) can result in numerous diseases that adversely affect health. Recent studies further elucidate the mechanistic details of the different forms of NETosis and their common end structure, as NETs were constantly found to contain DNA, modified histones and cytotoxic enzymes. In fact, emerging evidence reveal that the post translational modifications (PTMs) of histones in neutrophils have a critical role in regulating neutrophil death. Histone citrullination is shown to promote a rapid form of NET formation independent of NADPH oxidase (NOX), which relies on calcium influx. Interestingly, few studies suggest an association between histone citrullination and other types of PTMs to control cell survival and death, such as histone methylation. Even more exciting is the finding that histone acetylation has a biphasic effect upon NETosis, where histone deacetylase (HDAC) inhibitors promote baseline, NOX-dependent and -independent NETosis. However, increasing levels of histone acetylation suppresses NETosis, and to switch neutrophil death to apoptosis. Interestingly, in the presence of NETosis-promoting stimuli, high levels of HDACis limit both NETosis and apoptosis, and promote neutrophil survival. Recent studies also reveal the importance of the PTMs of neutrophils in influencing numerous pathologies. Histone modifications in NETs can act as a double-edged sword, as they are capable of altering multiple types of neutrophil death, and influencing numerous NET-mediated diseases, such as acute lung injury (ALI), thrombosis, sepsis, systemic lupus erythematosus, and cancer progression. A clear understanding of the role of different PTMs in neutrophils would be important for an understanding of the molecular mechanisms of NETosis, and to appropriately treat NETs-mediated diseases., Competing Interests: The authors declare that they have no conflict of interest related to this manuscript.

Neutrophil extracellular traps (NETs), a unique DNA framework decorated with antimicrobial peptides, have been in the scientific limelight for their role in a variety of pathologies ranging from cystic fibrosis to cancer. The formation of NETs, as well as relevant regulatory mechanisms, physiological factors, and pharmacological agents have not been systematically discussed in the context of their beneficial and pathological aspects. Novel forms of NET formation including vital NET formation continue to be uncovered, however, there remain fundamental questions around established mechanisms such as NADPH-oxidase (Nox)-dependent and Nox-independent NET formation. Whether NET formation takes place in the tissue versus the bloodstream, internal factors (e.g. reactive oxygen species (ROS) production and transcription factor activation), and external factors (e.g. alkaline pH and hypertonic conditions), have all been demonstrated to influence specific NET pathways. Elements of neutrophil biology such as transcription and mitochondria, which were previously of unknown significance, have been identified as critical mediators of NET formation through facilitating chromatin decondensation and generating ROS, respectively. While promising therapeutics inhibiting ROS, transcription, and gasdermin D are being investigated, neutrophil phagocytosis plays a critical role in host defense and any therapies targeting NET formation must avoid impairing the physiological functions of these cells. This review summarizes what is known in the many domains of NET research, highlights the most relevant challenges in the field, and inspires new questions that can bring us closer to a unified model of NET formation.

Identifying novel anti-cancer drugs is important for devising better cancer treatment options. In a series of studies designed to identify novel therapeutic compounds, we recently showed that a C-20 fatty acid (12,15-epoxy-13,14-dimethyleicosa-12,14-dienoic acid, a furanoic acid or F-6) present in the lipid fraction of the secretions of the Arabian Gulf catfish skin ( Arius bilineatus Val. ; AGCS) robustly induces neutrophil extracellular trap formation. Here, we demonstrate that a lipid mix (Ft-3) extracted from AGCS and F-6, a component of Ft-3, dose dependently kill two cancer cell lines (leukemic K-562 and breast MDA MB-231). Pure F-6 is approximately 3.5 to 16 times more effective than Ft-3 in killing these cancer cells, respectively. Multiplex assays and network analyses show that F-6 promotes the activation of MAPKs such as Erk, JNK, and p38, and specifically suppresses JNK-mediated c-Jun activation necessary for AP-1-mediated cell survival pathways. In both cell lines, F-6 suppresses PI3K-Akt-mTOR pathway specific proteins, indicating that cell proliferation and Akt-mediated protection of mitochondrial stability are compromised by this treatment. Western blot analyses of cleaved caspase 3 (cCasp3) and poly ADP ribose polymerase (PARP) confirmed that F-6 dose-dependently induced apoptosis in both of these cell lines. In 14-day cell recovery experiments, cells treated with increasing doses of F-6 and Ft-3 fail to recover after subsequent drug washout. In summary, this study demonstrates that C-20 furanoic acid F-6, suppresses cancer cell proliferation and promotes apoptotic cell death in leukemic and breast cancer cells, and prevents cell recovery. Therefore, F-6 is a potential anti-cancer drug candidate.

Acetylation is an important post translational modification of histone that plays a role in regulation of physiological and pathological process in the body. We have recently shown that the inhibition of histone deacetylases (HDAC) by low concentrations of HDAC inhibitors (HDACis), belinostat (up to 0.25 µM) and panobinostat (up to 0.04 µM) promote histone acetylation (e.g., AcH4) and neutrophil extracellular trap formation (NETosis). Clinical use of belinostat and panobinostat often leads to neutropenia and the in vivo concentrations vary with time and tissue locations. However, the effects of different concentrations of these HDACis on neutrophil death are not fully understood. We considered that increasing concentrations of belinostat and panobinostat could alter the type of neutrophil death. To test this hypothesis, we treated human neutrophils with belinostat and panobinostat in the presence or absence of agonists that promote NOX-dependent NETosis (phorbol myristate acetate or lipopolysaccharide from Escherichia coli 0128) and NOX-independent NETosis (calcium ionophores A23187 or ionomycin from Streptomyces conglobatus ). Increasing concentrations of HDACis induced histone acetylation in a dose-dependent manner. ROS analyses showed that increasing concentrations of HDACis, increased the degree of NOX-derived ROS production. Higher levels (>1 µM belinostat and >0.2 µM panobinostat) of AcH4 resulted in a significant inhibition of spontaneous as well as the NOX-dependent and -independent NETosis. By contrast, the degree of neutrophil apoptosis significantly increased, particularly in non-activated cells. Collectively, this study establishes that increasing concentrations of belinostat and panobinostat initially increases NETosis but subsequently reduces NETosis or switches the form of cell death to apoptosis. This new information indicates that belinostat and panobinostat can induce different types of neutrophil death and may induce neutropenia and regulate inflammation at different concentrations.

Genetic defects in cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene cause CF. Infants with CFTR mutations show a peribronchial neutrophil infiltration prior to the establishment of infection in their lung. The inflammatory response progressively increases in children that include both upper and lower airways. Infectious and inflammatory response leads to an increase in mucus viscosity and mucus plugging of small and medium-size bronchioles. Eventually, neutrophils chronically infiltrate the airways with biofilm or chronic bacterial infection. Perpetual infection and airway inflammation destroy the lungs, which leads to increased morbidity and eventual mortality in most of the patients with CF. Studies have now established that neutrophil cytotoxins, extracellular DNA, and neutrophil extracellular traps (NETs) are associated with increased mucus clogging and lung injury in CF. In addition to opportunistic pathogens, various aspects of the CF airway milieux (e.g., airway pH, salt concentration, and neutrophil phenotypes) influence the NETotic capacity of neutrophils. CF airway milieu may promote the survival of neutrophils and eventual pro-inflammatory aberrant NETosis, rather than the anti-inflammatory apoptotic death in these cells. Degrading NETs helps to manage CF airway disease; since DNAse treatment release cytotoxins from the NETs, further improvements are needed to degrade NETs with maximal positive effects. Neutrophil-T cell interactions may be important in regulating viral infection-mediated pulmonary exacerbations in patients with bacterial infections. Therefore, clarifying the role of neutrophils and NETs in CF lung disease and identifying therapies that preserve the positive effects of neutrophils, while reducing the detrimental effects of NETs and cytotoxic components, are essential in achieving innovative therapeutic advances., Competing Interests: The authors declare no conflict of interest.

Neutrophils undergo a unique form of cell death to generate neutrophil extracellular traps (NETs). It is well established that citrullination of histones (e.g., CitH3) facilitates chromatin decondensation during NET formation (NETosis), particularly during calcium-induced NETosis that is independent of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activation. However, the importance of other forms of histone modifications in NETosis has not been established. We considered that acetylation of histones would also facilitate NETosis. To test this hypothesis, we induced NOX-dependent NETosis in human neutrophils with phorbol myristate acetate or lipopolysaccharide (from Escherichia coli 0128), and NOX-independent NETosis with calcium ionophores A23187 or ionomycin (from Streptomyces conglobatus ) in the presence or absence of two pan histone deacetylase inhibitors (HDACis), belinostat and panobinostat (within their half maximal inhibitory concentration (IC50) range). The presence of these inhibitors increased histone acetylation (e.g., AcH4) in neutrophils. Histone acetylation was sufficient to cause a significant increase (~20%) in NETosis in resting neutrophils above baseline values. When acetylation was promoted during NOX-dependent or -independent NETosis, the degree of NETosis additively increased (~15⁻30%). Reactive oxygen species (ROS) production is essential for baseline NETosis (mediated either by NOX or mitochondria); however, HDACis did not promote ROS production. The chromatin decondensation step requires promoter melting and transcriptional firing in both types of NETosis; consistent with this point, suppression of transcription prevented the NETosis induced by the acetylation of histones. Collectively, this study establishes that histone acetylation (e.g., AcH4) promotes NETosis at baseline, and when induced by both NOX-dependent or -independent pathway agonists, in human neutrophils. Therefore, we propose that acetylation of histone is a key component of NETosis., Competing Interests: The authors declare that they have no conflict of interest.

Various biomolecules induce neutrophil extracellular trap (NET) formation or NETosis. However, the effect of fatty acids on NETosis has not been clearly established. In this study, we focused on the NETosis-inducing ability of several lipid molecules. We extracted the lipid molecules present in Arabian Gulf catfish ( Arius bilineatus , Val) skin gel, which has multiple therapeutic activities. Gas chromatography⁻mass spectrometry (GC-MS) analysis of the lipid fraction-3 from the gel with NETosis-inducing activity contained fatty acids including a furanoid F-acid (F6; 12,15-epoxy-13,14-dimethyleicosa-12,14-dienoic acid) and common long-chain fatty acids such as palmitic acid (PA; C16:0), palmitoleic acid (PO; C16:1), stearic acid (SA; C18:0), and oleic acid (OA; C18:1). Using pure molecules, we show that all of these fatty acids induce NETosis to different degrees in a dose-dependent fashion. Notably, F6 induces a unique form of NETosis that is rapid and induces reactive oxygen species (ROS) production by both NADPH oxidase (NOX) and mitochondria. F6 also induces citrullination of histone. By contrast, the common fatty acids (PA, PO, SA, and OA) only induce NOX-dependent NETosis. The activation of the kinases such as ERK (extracellular signal-regulated kinase) and JNK (c-Jun N-terminal kinase) is important for long-chain fatty acid-induced NETosis, whereas, in F-acid-induced NETosis, Akt is additionally needed. Nevertheless, NETosis induced by all of these compounds requires the final chromatin decondensation step of transcriptional firing. These findings are useful for understanding F-acid- and other fatty acid-induced NETosis and to establish the active ingredients with therapeutic potential for regulating diseases involving NET formation.